We propose to develop an innovative high-expression CHO cell based platform for rapid Influenza vaccine production, which will have the ability to complete a 12 week vaccine production cycle, leading to a drastic reduction in response time to Influenza outbreak. This system will have advantages over the traditional approach to vaccine production in chicken eggs in that it can more feasibly meet the rapid surge capacity needs that would be required during a pandemic outbreak. Traditional methods of generating Influenza vaccines in embryonated chicken eggs fall short of the needs during a pandemic because of lengthy times required for scale-up, high costs, limited quantities of specialized eggs and potential risks of contamination. Cell-culture based vaccines are potentially a more reliable, flexible, and easily scalable method of producing vaccines; however, cell lines available for efficient large-scale vaccine production are limited. The approach is to integrate two novel technologies for creating a highly efficient, rapid vaccine production system within mammalian cells. The first technology, developed by FluGen, increases the number of surface bound influenza virus receptors for expanding the amount of virus that can infect the cell. The second technology, developed by CDI Bioscience, is incorporated into a high-expression mammalian host cell line (PACE) that, when activated, will be capable of more than doubling the expression of the surface receptors of the FluGen technology. We will achieve the objectives of the project by pursuing three specific aims over a three year period: (Aim 1) Develop a PACE cell line that stably expresses FluGen surface receptors on cell membrane; Three to four high-expressing PACE clonal lines stably expressing FluGen surface receptors on cell membrane will be developed. These clonal cell lines will be considered suitable for further characterization if they (a) show over 2-fold FluGen surface receptors expression upon RP shift; and (b) produce higher viral titers when infected with H1N1 (A/PR/8/34 strain) virus, as compared to the PACE cells that are not engineered with the FluGen surface receptors. (Aim 2) Develop bioprocess protocols for influenza virus production in FluGen surface receptors expressing PACE cells in shake flasks; We will determine the best CD medium for cell growth and culture viability, and the optimal VCD for induction of RP shift(R). We will then determine the best medium/feed/supplements combination that achieves: (a) at least 2-fold higher expression of FluGen surface receptors upon induction of RP shift(R). (2) Virus titers of at least 107-108 pfu/ml. (Aim 3) Develop scalable bioprocess protocols for influenza virus production in bench top and industrial bioreactors (50-100L); Adapted cells and optimal conditions from Aim 2 will be used in small-scale bench top bioreactors to determine the best cell growth and induction conditions in order to achieve the highest viral titer in an industrial environment. Subsequently, we will develop scale up strategy for optimal influenza virus production in 50-100L bioreactor. Narrative Traditional methods of generating Influenza vaccines in embryonated chicken eggs fall short of the needs during a pandemic outbreak. This project combines two novel technologies for creating a highly efficient, rapid vaccine production system within mammalian cells, allowing completion of a 12 week vaccine production cycle. Achieving the objectives of this project will form a major milestone towards leading to a drastic reduction in response time to Influenza outbreak.